Chromosomal multidrug resistance (MDR) in bacteria is a serious clinical problem. Our studies have shown that Escherichia coli becomes resistant to a variety of antibiotics, organic solvents and superoxides when the activities of any of three paralogous, but differently regulated, transcriptional activators, MarA, SoxS and Rob, are increased. These activators bind a sequence called the marbox which lies upstream of the promoters of a set of at least 40 chromosomal genes called the marA/soxS/rob regulon. The expression of three proteins upregulated by MarA, SoxS and Rob are particularly important for MDR in E. coli. 1) TolC, encodes an outer membrane protein that interacts with 2) AcrB, one of nine different inner membrane efflux pumps and 3) AcrA, a cognate periplasmic protein. This AcrAB-TolC tripartite complex forms a trans-periplasmic channel for extruding antibiotics directly out of the cell into the environment. We have recently shown that TolC and several other pumps reduce cellular stress by expelling toxic cellular metabolites. Metabolomic studies are currently underway (with K. van de Wetering, Amsterdam) to identify these metabolites. It is surprising that the AcrAB-TolC pump is able to extrude a wide variety of substances, yet not injure the cell by losing essential metabolites. We are now investigating the basis of this discrimination by looking for mutations in acrB that convert it into a gene that is inhibitory for cellular growth. The idea is that the mutant acrB will encode a protein that has lost the ability to discriminate between the toxic compounds entering the cell from useful compounds present in the cell and thereby have reduced growth when the gene is overexpressed. We believe that this may allow us to define the amino acids of AcrB that are responsible for this discrimination and will suggest new targets for drug discovery that will counter MDR. Thiazolides are effective antibiotics against intracellular protozoans, anaerobic or microaerophilic bacteria, viruses and tumor cells. Together with N. Muller (Berne), we have found that aerobically growing E. coli are resistant to these compounds but tolC mutants are susceptible. This suggests that TolC-mediated efflux is responsible for the otherwise observed thiazolide resistance and creates good possibilities for discovery of the mechanism of action of these important drugs.